1. Field of the Invention
The present invention relates generally to measuring collagen content in tissues. More particularly, it concerns the measurement of collagen content using HPLC technology. In specific embodiments, the invention concerns measuring collagen content in healing wounds.
2. Description of Related Art
Collagen protein is made of polypeptide chains composed of a repeated sequence of amino acids primarily consisting of hydroxyproline (Hyp), glycine (Gly), and proline (Pro). Collagen is one of the most predominant proteins found in the human body, comprising about 80-85% of the extracellular matrix (ECM) in the dermal layer of normal (non-wounded) skin tissue. Evidence suggests that collagen's unique characteristics provide normal skin tissue with tensile strength, integrity, and structure. G. S. Schultz, et al., Extracellular matrix: review of its roles in acute and chronic wounds, World Wide Wounds, (August 2005) on the world wide web at worldwidewounds.com/2005/august/Schultz/Extrace-Matric-Acute-Chronic-Wounds.html. Since generation of tensile strength is an important component of successful wound healing and because collagen is the main protein involved in the generation of tensile strength, accurate collagen measurements may be predictive of successful wound healing.
Various analytical methods commonly used in biochemistry and analytical chemistry may be appropriate for quantifying collagen, such as spectrophotometry and gas chromatography. H. Inoue, et al., J. of Chromatography B, 724:221-230 (1999). Most currently available spectrophotometer calorimetric methods for measuring hydroxyproline are modifications of the 1950 Neuman and Logan or Stegemann methods, which may be sensitive and accurate, but are also cumbersome and problematic. H. Stegemann and K. Stalder, Clinica Chemical Acta, 18:267-273 (1967); B. R. Switzer and G. K. Summer, Analytical Biochemistry 39:487-491 (1971).
Another common analytical method is high pressure liquid chromatography (HPLC). HPLC operates by forcing a sample through a specially packed column by pumping a liquid, to which the sample is added, through the column at high pressure. The material packed inside the column is referred to as the stationary or adsorbent phase, and is usually finely ground powders or gels. The liquid, which is referred to as the mobile phase, is commonly an organic and/or buffered solution. Substances within the sample that are separated during chromatography for study are commonly called analytes. A chromatogram, the visual output of a chromatograph, displays different peaks or patterns corresponding to different components (such as analytes) of the separated mixture. HPLC generated data allows an analyst to determine if there are interfering or co-eluting peaks, and exactly where these peaks originate. The peaks generated by HPLC may be analyzed for separation, whereas it is not possible to determine if results from a spectrophotometer are due to contaminating compounds.
Known methods of using HPLC to measure collagen require specialized equipment such as fluorescence detectors or highly specialized columns, which can increase the cost and/or complexity of analysis, potentially rendering the procedure prohibitive. H. Inoue, et al; J. of Chromatography B, 757:369-373 (2001); D. A. Martinez et al., Diabetes Res. and Clinical Practice, 591-9 (2003); F. A. Vázquez-Ortíz, et al., J of Liquid Chromatography & Related Tech., 27, 17 2771-2780 (2004). Inoue, et al. describe a method of using HPLC to determine human serum levels of prolyl dipeptides, proline, and hydroxyproline as an indicator of diseases involving collagen metabolism. This method requires tandem HPLC columns and monitoring at two different emission wavelengths. Martinez et al. analyzed hydroxyproline utilizing a reverse phase HPLC method with the Waters Pico-Tag® column, which is a dedicated column specially packed for amino acid analysis. By quantifying hydroxyproline and hydroxylysylpyridinoline cross links, Martinez et al. an index of collagen content in pig left ventricle was obtained, but collagen content was not quantified. Vázquez-Ortíz, et al. described a method of determining collagen concentration in meat products such as bologna. This method involves measuring hydroxyproline content using reverse phase HPLC and a fluorichrom detector.